Genome integrity is essential for human health and the viability of all species. A major source of genome instability is the stimulation of homologous recombination (HR) that occurs when replication forks arrest at lesions in the DNA template. Although cell-cycle checkpoints and DNA repair enzymes typically repair such lesions with high fidelity, defects in these processes are associated with human disease and cancer. One such disease, Bloom Syndrome, arises from defects in BLM, a member of RecQ family of DNA helicases. BLM acts together with DNA topoisomerase III and a newly-identified subunit, Rmi1, to suppress sister chromatid exchange. The fundamental nature of this complex is underscored by its conservation in lower eukaryotes such as budding yeast. As in humans, loss of the homologous Sgs1-Top3-Rmi1 (STR) complex in yeast results in genome instability and enhanced sensitivity to DNA damage. In this project we will exploit the biochemistry and genetics of yeast to determine how post-translational modification of proteins with SUMO regulates the DNA repair pathways that the cell uses in the absence of STR. In Aim 1 we will determine the biochemical and genetic function of the Slx5-Slx8 Ub ligase which is essential for viability in the absence of STR. We will test the hypothesis that Slx5-Slx8 activity leads to the proteasomal destruction of poly- sumoylated proteins. In-vivo and in-vitro assays will be used to determine how the ubiquitination of sumoylated proteins by Slx5-Slx8 suppresses genome instability. This will involve identifying relevant in-vivo target proteins as well as characterizing the enzyme's preferred substrate which may be a specific form of poly-SUMO chains. In Aim 2 we will examine the function of Wss1 which is a new player in the control of sumoylation and genome stability. We will determine whether Wss1 is a SUMO isopeptidase using in-vitro assays and a variety of sumoylated test substrates. In Aim 3 we will determine the broader significance of poly- SUMO conjugates that arise in certain DNA repair mutants. We will examine the phenotype of such mutants when they are unable to polymerize SUMO chains, and identify additional DNA repair mutants that give rise to poly-sumoylated proteins. We will also examine the role of the Ulp2 isopeptidase in genome maintenance by determining how a mutant allele of ULP2 suppresses the lethality of sgs1 slx5 mutants. PUBLIC HEALTH RELEVANCE: Genome integrity is essential for the health and viability of all organisms, including humans. For example, patients with Bloom Syndrome (BS) lack the BLM protein and suffer from genome instability that eventually leads to cancer. This project seeks to characterize the DNA repair pathways that operate in the absence of BLM using yeast as a model system. The project will exploit well-known features of this model system to determine role of protein modification by SUMO and to characterize alternative repair pathways that function in the absence of this modification. Thus, this research will provide new understanding about the factors and genetic pathways that maintain genome stability in normal and BS cells.